US20020037581A1

US20020037581A1 - Extracellular serine protease

Info

Publication number: US20020037581A1
Application number: US09/796,294
Authority: US
Inventors: Timothy O'Brien; Lowell Underwood
Original assignee: UAB Research Foundation
Current assignee: UAB Research Foundation; University of Arkansas for Medical Sciences
Priority date: 1997-08-21
Filing date: 2001-02-28
Publication date: 2002-03-28
Also published as: US7157084B2

Abstract

The present invention provides a DNA encoding a novel extracellular serine protease termed Tumor Antigen Derived Gene-14 (TADG-14) which is overexpressed in ovarian, breast and colon carcinoma samples. Also provided are vector and host cells capable of expressing the DNA of the present invention, as well as the uses of the DNA and protein of the present invention.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a continuation-in-part of U.S. application Ser. No. 09/618,259 filed Jul. 18, 2000, which is a continuation-in-part of U.S. application Ser. No. 09/137,944 filed Aug. 21, 1998, which is a continuation-in-part of U.S. application Ser. No. 08/915,659 filed Aug. 21, 1997.[0001]

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cellular biology and the diagnosis of neoplastic disease. More specifically, the present invention relates to a novel extracellular serine protease termed Tumor Antigen Derived Gene-14 (TADG-14).

2. Description of the Related Art

Serine proteases comprise a family of protein degrading enzymes that serve a host of biological functions including activation of blood coagulation cascades, activation of growth and angiogenic factors and degradation of extracellular matrix components (1-4). In recent years, aberrant expression of serine proteases, such as plasminogen activator have been shown to correlate positively with the invasiveness and metastatic potential of tumor cells (3, 5-6). Presumably, this occurs by increasing the ability of the tumors to degrade extracellular matrix components either directly or indirectly through the proteolytic activation of other zymogenic proteases. More significantly, the serine protease known as the prostate specific antigen (PSA) has been used successfully as a tumor marker for the early diagnosis of prostate cancer due to its abnormal prevalence in the peripheral blood of these patients (7). Serine proteases play important roles in the cascade of events involved in the malignant process, and at least for prostate cancer, they provide sufficient signal to allow detection of early disease.

The prior art is deficient in the lack of effective means of screening to identify proteases overexpressed in carcinoma. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention discloses a 1343 base pairs long TADG-14 cDNA (SEQ ID No: 6) which encodes a 260 amino acid protein (SEQ ID No: 7) overexpressed in carcinoma. The availability of the TADG-14 gene opens the way for a number of studies that can lead to various applications.

In one embodiment of the present invention, there is provided a DNA encoding a TADG-14 protein selected from the group consisting of: (a) isolated DNA having the sequence of SEQ ID No. 6; (b) isolated DNA which is complementary to the isolated DNA of (a); and (c) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code.

In another embodiment of the present invention, there is provided a vector capable of expressing the DNA of the present invention, as well as host cell transfected with the vector that express a TADG-14 protein. Additionally embodied is a vector in 5 which the TADG-14 DNA is positioned in reverse orientation relative to the regulatory elements such that TADG-14 antisense DNA is produced.

In yet another embodiment of the present invention, there is provided an isolated and purified TADG-14 protein coded for by DNA selected from the group consisting of: (a) isolated DNA having the sequence of SEQ ID No. 6; and (b) isolated DNA differing from the isolated DNA of (a) in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-14 protein.

In still yet another embodiment of the present invention, there is provided a method for detecting TADG-14 mRNA in a biological sample, comprising the steps of: (a) contacting said sample with a probe specific for TADG-14, wherein said probe comprises of sequence complementary to SEQ ID No. 6; and (b) detecting binding of the probe to TADG-14 mRNA in the sample. Generally, the sample is a biological sample from blood, interstitial fluid, ascites fluid, tumor tissue biopsy or circulating tumor cells. Preferably, the biological sample is from an individual; and typically, the individual is suspected of having cancer.

In yet another embodiment of the present invention, there is provided a kit for detecting TADG-14 mRNA, comprising: an oligonucleotide probe specific for TADG-14. A label for detection is further embodied in the kit.

In yet another embodiment of the present invention, there is provided a method of detecting TADG-14 protein in a biological sample, comprising the steps of: (a) contacting the sample with an antibody which is specific for TADG-14 or a fragment thereof; and (b) detecting binding of the antibody to TADG-14 protein in the sample. Similarly, the present invention also embodies a kit for detecting TADG-14 protein, comprising an antibody specific for TADG-14 protein or a fragment thereof. Means for detection of the antibody is further embodied in the kit.

In yet another embodiment of the present invention, there is provided an antibody specific for the TADG-14 protein or a fragment thereof.

In another embodiment of the present invention, there is provided a method of screening for compounds that inhibit TADG-14 protease activity, comprising the steps of: (a) contacting a sample comprising TADG-14 protein with a compound; and (b) assaying for TADG-14 protease activity. Typically, a decrease in the TADG-14 protease activity in the presence of the compound relative to TADG-14 protease activity in the absence of the compound is indicative of a compound that inhibits TADG-14 protease activity.

In still yet another embodiment of the present invention, there is provided methods of inhibiting expression of TADG-14 in a cell with TADG-14 antisense DNA or TADG-14-specific antibody. Generally, the inhibition of TADG-14 expression is for treating cancer.

In another embodiment of the present invention, there is provided a method of targeted therapy to an individual, comprising the step of: (a) administering a compound containing a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-14.

In another embodiment of the present invention, there is provided a method of diagnosing cancer in an individual, comprising the steps of: (a) obtaining a biological sample from an individual; and (b) detecting TADG-14 in the sample, wherein the presence of TADG-14 in the sample is indicative of the presence of carcinoma in the individual.

In yet another embodiment of the present invention, there is provided a method of vaccinating an individual against TADG-14 protein, comprising the step of (a) inoculating an individual with a TADG-14 protein or fragment thereof which lacks TADG-14 protease activity. Typically, inoculation with the TADG-14 protein or fragment thereof elicits an immune response in the individual, thereby vaccinating the individual against TADG-14. Generally, the individual has cancer, is suspected of having cancer or is at risk of getting cancer. Preferably, the TADG-14 fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, or 64.

In another embodiment of the present invention, there is provided a method of producing activated immune cells directed toward TADG-14, comprising the steps of exposing immune cells to a TADG-14 protein or fragment thereof which lacks TADG-14 protease activity. Usually, exposure to the TADG-14 protein or fragment thereof activates the immune cells, thereby producing activated immune cells directed toward TADG-14. Generally, the immune cells are B cells, T cells or dendritic cells. Preferably, the dendritic cells are isolated from an individual prior to exposure to a TADG-14 protein or fragment thereof, and then reintroduced into the individual subsequent to the exposure. Typically, the individual has cancer, is suspected of having cancer or is at risk of getting cancer. Preferably, the TADG-14 fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, or 64.

In another embodiment of the present invention, there is provided an immunogenic composition, comprising an immunogenic fragment of a TADG-14 protein and an appropriate adjuvant. Preferably, the TADG-14 fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, or 64.

In another embodiment of the present invention, there is provided an oligonucleotide having a sequence complementary to SEQ ID No. 6, as well as a composition comprising the oligonucleotide and a physiologically acceptable carrier.

Additionally, there is provided a method of treating a neoplastic state in an individual in need of such treatment, comprising the step of (a) administering to the individual an effective dose of the above- described oligonucleotide.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope. patients, may prove to be a useful tool for early detection of ovarian cancer, especially the most prevalent serous cystadenocarcinoma subtype. [0025]

EXAMPLE 12

Peptide ranking analysis for vaccine candidates [0026]
For vaccine or immune stimulation, individual 9-mers to 11-mers of the TADG-14 protein were examined to rank the binding of individual peptides to the top 8 hapIotypes in the general population (Parker et al., (1994)). Table 2 shows the peptide ranking based upon the predicted half-life of each peptide's binding to a particular HLA allele. A larger half-life indicates a stronger association with that peptide and the particular HLA molecule. The TADG-14 peptides that strongly bind to an HLA allele are putative immunogens, and are used to innoculate an individual against TADG-14. sequence for initiation of translation and the polyadenylation signal, respectively. The TADG-14 protein sequence contains a secretion signal sequence near its amino terminus. The stop codon is represented by the (*) symbol. [0027]
FIG. 3B shows the amino acid sequence of TADG-14 compared to human glandular kallikrein (hHk2, accession # P06870), human PSA (hPSA, accession # P07288), mouse neuropsin (mNeur, accession # D30785) and human Protease M (hProM, accession # U62801) using the GCG PILEUP program (REF). The positions of the residues of the catalytic triad are marked Y. [0028]
FIG. 4 shows a comparison of the amino acid sequence of TADG-14's catalytic domains. [0029]
FIG. 5 shows the TADG-14 quantitative PCR. FIG. 5A shows the typical results of a TADG-14 quantitative PCR experiment. The reaction products were electrophoresed through a 2% agarose TAE gel and stained with ethidium bromide. In this figure, the 454-bp band represents the β-tubulin product and the 230-bp band represents the TADG-14 product. The radiolabeled PCR products were quantitated. [0030]
FIG. 5B shows the overexpression of TADG-14. As determined by the student's t test, TADG-14 mRNA expression levels were significantly elevated in LMP tumors (*, P=0.05) and carcinomas (P<0.0001) compared to levels found in normal ovary. Individual cases are represented in a scatter plot. This is indicative of heterogeneity of TADG-14 expression among these tumor samples. [0031]
FIG. 6 shows the TADG-14 expression in tumors and cell lines. [0032]
FIG. 7 shows Western blot analysis of TADG-14. Polyclonal antibodies were generated by immunization of rabbits with one of three poly-lysine linked multiple antigen peptides derived from the deduced amino acid sequence of TADG-14. For Western blot analysis, approximately 20 ug of MDA-MB-435S and HeLa cell lysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDF at 100 V for 40 minutes at 4 C. The blot was blocked overnight in Tris-buffered saline (TBS), pH 7.8 containing 0.2% non-fat milk. Primary antibody was added to the membrane at a dilution of 1:100 in 0.2% milk/TBS and incubated for 2 hours at room temperature. The blot was washed and incubated with 1:3000 dilution of alkaline-phosphatase conjugated goat and anti-rabbit IgG antibody (Bio-Rad) for one hour at room temperature. The blot was washed and incubated with a chemiluminescent substrate (Bio-Rad) before a 10-second exposure to X-ray film for visualization. [0033]
FIG. 8 shows immunohistochemistry of TADG-14. [0034]
Staining was with the TADG-14-1 antibody for normal ovary, two serous carcinomas, mucinous carcinoma, endometrioid carcinoma and clear cell carcinoma of the ovary (FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D, FIG. 8E and FIG. 8F, respectively). No staining was observed in normal ovary. The serous carcinoma shown in FIG. 8B has TADG-14 most strongly associated with the surface of the tumor, while in the serous tumor in FIG. 8C, TADG-14 was found in a granular form in an apparent secretion pathway. In mucinous carcinoma TADG-14 appears to be most highly expressed along the invasive front of the tumor. TADG-14 was secreted into the lumen of the glandular structure formed by the endometrioid carcinoma in FIG. 8E. The clear cell carcinoma stained in FIG. 8F shows diffuse staining throughout all tumor cells. [0035]

DETAILED DESCRIPTION OF THE INVENTION

All serine proteases contain conserved histidine, aspartate and serine residues that are necessary for enzymatic activity. To identify the expressed serine proteases in carcinoma, degenerate oligodeoxynucleotide primers designed to the conserved amino acid sequences surrounding the invariant His and Ser residues of the catalytic triad (8) were used in PCR reactions with cDNA from either normal ovarian tissue or ovarian carcinoma as the template. PCR products of the appropriate size were subcloned into T-vector and sequenced. Previously, this strategy has proved successful in identifying the serine proteases hepsin and stratum corneum chymotryptic enzyme (SCCE) which have been shown to be expressed at abnormally high levels in ovarian carcinoma (9, 10). [0036]
Homology searches revealed that one of the subclones obtained from ovarian carcinoma represented a novel 406 base pair (bp) sequence that has significant sequence similarity to other known proteases including mouse neuropsin, human glandular kallikrein and human PSA. The complete cDNA for this novel sequence was cloned and found to encode a trypsin like serine protease, named TADG-14. The TADG-14 cDNA is 1343 base pairs long (SEQ ID No: 6) and encoding for a 260 amino acid protein (SEQ ID No: 7). [0037]
The availability of the TADG-14 gene opens the way for a number of studies that can lead to various applications. More importantly, the TADG-14 transcript was found to be highly expressed in a majority of ovarian tumors but not expressed by normal ovarian tissue. High level expression of TADG-14 appears to be restricted to tumors, and this protease appears to be secreted in a manner that would suggest a possible role in invasion and metastasis. Moreover, due to the extracellular nature of this enzyme, it may be possible to exploit its expression as a diagnostic tool for ovarian cancer. [0038]
In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See e.g., Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D.N. Glover ed. 1985); “Oligonucleotide Synthesis” (M.J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription and Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal Cell Culture” [R.I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984). [0039]
As used herein, the term “cDNA” shall refer to the DNA copy of the mRNA transcript of a gene. [0040]
As used herein, the term “derived amino acid sequence” shall mean the amino acid sequence determined by reading the triplet sequence of nucleotide bases in the cDNA. [0041]
The amino acid described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. [0042]
As used herein the term “screening a library” shall refer to the process of using a labeled probe to check whether, under the appropriate conditions, there is a sequence complementary to the probe present in a particular DNA library. In addition, “screening a library” could be performed by PCR. [0043]
As used herein, the term “PCR” refers to the polymerase chain reaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, as well as other improvements now known in the art. [0044]
A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, [0045] inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide [0046] in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
The term “oligonucleotide”, as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. [0047]
The term “primer” as used herein refers to an oligonucleotide occurring naturally as in a purified restriction digest or produced synthetically which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. [0048]
The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence or hybridize therewith and thereby form the template for the synthesis of the extension product. [0049]
A cell has been “transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth [0050] in vitro for many generations.
As used herein, the term “host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. A recombinant DNA molecule or gene which encodes a human TADG-14 protein of the present invention can be used to transform a host using any of the techniques commonly known to those of ordinary skill in the art. Especially preferred is the use of a vector containing coding sequences for the gene which encodes a human TADG-14 protein of the present invention for purposes of prokaryote transformation. [0051]
Prokaryotic hosts may include [0052] E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells.
A “heterologous' region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. [0053]
In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth. [0054]
The invention includes a substantially pure DNA encoding a TADG-14 protein, containing a sequence of at least 1 5 consecutive nucleotides (preferably 20, more preferably 30, even more preferably 50, and most preferably all) of the region from [0055] nucleotides 1 to 1343 of the nucleotides listed in SEQ ID NO: 6. The protein encoded by the DNA of this invention may share at least 80% sequence identity (preferably 85%, more preferably 90%, and most preferably 95%) with the amino acids listed in SEQ ID NO: 7. More preferably, the DNA includes the coding sequence of the nucleotides of SEQ ID NO: 6, or a degenerate variant of such a sequence.
“Substantially pure DNA” is DNA that is part of a milieu in which the DNA does not naturally occurs. The DNA can be obtained by virtue of separation (partial or total purification) of some or all of the molecules of that milieu, or by virtue of alteration of sequences that flank the claimed DNA. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, an autonomously replicating plasmid or virus, the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, e.g., a fusion protein. Also included is a recombinant DNA which includes a portion of the nucleotides listed in SEQ ID NO: 6 which encodes an alternative splice variant of TADG-14. [0056]
The present invention encompasses DNA that have at least about 70% sequence identity to the coding sequence of the nucleotides listed in SEQ ID NO: 6, preferably at least 75% (e.g. at least 80%); and most preferably at least 90%. The identity between two sequences is a direct function of the number of matching or identical positions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then they are identical at that position. For example, if 7 positions in a [0057] sequence 10 nucleotides in length are identical to the corresponding positions in a second 10-nucleotide sequence, then the two sequences have 70% sequence identity. The length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
The invention also includes DNA that hybridizes at high stringency to a probe containing at least 15 consecutive nucleotides of SEQ ID NO: 6. The probe to which the DNA of the invention hybridizes preferably consists of a sequence of at least 20 consecutive nucleotides, more preferably 40 nucleotides, even more preferably 50 nucleotides, and most preferably 100 nucleotides or more (up to 100%) of the coding sequence of the nucleotides listed in SEQ ID NO: 6 or the complement thereof. Such a probe is useful for detecting expression of TADG-14 in a cell by a method including the steps of (a) contacting mRNA obtained from the cell with the labeled hybridization probe; and (b) detecting hybridization of the probe with the mRNA. [0058]
By “high stringency” is meant DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65° C. at a salt concentration of approximately 0.1×SSC, or the functional equivalent thereof. For example, high stringency conditions may include hybridization at about 42° C. in the presence of about 50% formamide; a first wash at about 65° C. with about 2×SSC containing 1% SDS; followed by a 5 second wash at about 65° C. with about 0.1×SSC. [0059]
The present invention further comprises a vector comprising a DNA sequence which encodes a human TADG-14 protein and said vector comprises in operable linkage: a) an origin of replication; b) a promoter; and c) a DNA sequence coding for said protein. Preferably, the vector of the present invention contains a portion of the DNA sequence shown in SEQ ID No: 6. [0060]
A “vector” may be defined as a replicable nucleic acid construct, e.g., a plasmid or viral nucleic acid. Vectors may be used to amplify and/or express nucleic acid encoding TADG-14 protein. An “expression vector” is a replicable construct in which a nucleic acid sequence encoding a polypeptide is operably linked to suitable control sequences capable of effecting expression of the polypeptide in a cell. The need for such control sequences will vary depending upon the cell selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter and/or enhancer, suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation. Methods which are well known to those skilled in the art can be used to construct expression vectors containing appropriate transcriptional and translational control signals. See for example, the techniques described in Sambrook et al., 1989, [0061] Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor Press, N.Y. A gene and its transcription control sequences are defined as being “operably linked” if the transcription control sequences effectively control the transcription of the gene. Vectors of the invention include, but are not limited to, plasmid vectors and viral vectors. Preferred viral vectors of the invention are those derived from retroviruses, adenovirus, adeno—associated virus, SV40 virus, or herpes viruses.
Further included in this invention are substantially pure TADG-14 proteins which are encoded at least in part by portions of SEQ ID NO: 7, e.g., products of alternative mRNA splicing or alternative protein processing events, or in which a section of TADG-14 sequence has been deleted. The fragment, or the intact TADG-14 polypeptide, may be covalently linked to another polypeptide, e.g. which acts as a label, a ligand or a means to increase antigenicity. [0062]
By a “substantially pure protein” is meant a protein which has been separated from at least some of those components which naturally accompany it. Typically, the protein is substantially pure when it is at least 60% by weight free from the proteins and other naturally-occurring organic molecules with which it is naturally associated [0063] in vivo. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure TADG-14 protein may be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a TADG-14 polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography such as immunoaffinity chromatography using an antibody specific for TADG-14, polyacrylamide gel electrophoresis, or HPLC analysis. A protein which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be, by definition, substantially free from its naturally associated components. Accordingly, substantially pure proteins include eukaryotic proteins synthesized in E. coli, other prokaryotes, or any other organism in which they do not naturally occur.
In addition to substantially full-length proteins, the invention also includes fragments (e.g., antigenic fragments) of the TADG-14 protein (SEQ ID No: 7). As used herein, “fragment,” as applied to a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments of the TADG-14 protein can be generated by methods known to those skilled in the art, e.g., by enzymatic digestion of naturally occurring or recombinant TADG-14 protein, by recombinant DNA techniques using an expression vector that encodes a defined fragment of TADG-14, or by chemical synthesis. The ability of a candidate fragment to exhibit a characteristic of TADG-14 (e.g., binding to an antibody specific for TADG-14) can be assessed by methods described herein. Purified TADG-14 or antigenic fragments of TADG-14 can be used to generate new antibodies or to test existing antibodies (e.g., as positive controls in a diagnostic assay) by employing standard protocols known to those skilled in the art. [0064]
Included in this invention are polyclonal or monoclonal antibodies generated by using TADG-14 or a fragment of TADG-14 as the immunogen in, e.g., rabbits. Standard protocols for monoclonal and polyclonal antibody production known to those skilled in this art are employed. The monoclonal antibodies generated by this procedure can be screened for the ability to identify recombinant TADG-14 cDNA clones, and to distinguish them from known cDNA clones. [0065]
The invention encompasses not only an intact polyclonal or monoclonal antibody, but also an immunologically-active antibody fragment that recognizes TADG-14, e.g., a Fab or (Fab)[0066] ₂fragment; an engineered single chain Fv molecule; or a chimeric antibody in which the antigen binding sites are derived from murine antibody while the remaining portions of the antibody are of human origin.
In one embodiment, the antibody, or a fragment thereof, may be linked to a toxin or to a detectable label, e.g. a radioactive label, non-radioactive isotopic label, fluorescent label, chemiluminescent label, paramagnetic label, enzyme label, or calorimetric label. Examples of suitable toxins include diphtheria toxin, [0067] Pseudomonas exotoxin A, ricin, and cholera toxin. Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase, etc. Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, etc.
Paramagnetic isotopes for purposes of [0068] in vivo diagnosis can also be used according to the methods of this invention. There are numerous examples of elements that are useful in magnetic resonance imaging. For discussions on in vivo nuclear magnetic resonance imaging, see, for example, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986) Magn. Reson. Med. 3, 336-340; Wolf, G. L., (1984) Physiol. Chem. Phys. Med. NMR 16, 93-95; Wesbey et al., (1984) Physiol. Chem. Phys. Med. NMR 16, 145-155; Runge et al., (1984) Invest. Radiol. 19, 408-415. Examples of suitable fluorescent labels include a fluorescein label, an isothiocyalate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an ophthaldehyde label, a fluorescamine label, etc. Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, a luciferase label, an aequorin label, etc.
Those of ordinary skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy et al., (1976) [0069] Clin. Chim. Acta 70, 1-31; and Schurs et al., (1977) Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dimaleimide method, the m—maleimidobenzyl—N—hydroxy—succinimide ester method. All of these methods are incorporated by reference herein.
As described herein, the invention provides a number of diagnostic advantages and uses. For example, the TADG-14 protein is useful in diagnosing cancer in different tissues since this protein is absent in highly proliferating cells. Antibodies (or antigen-binding fragments thereof) which bind to an epitope specific for TADG-14, are useful in a method of detecting TADG-14 protein in a biological sample for diagnosis of cancerous or neoplastic transformation. This method includes the steps of obtaining a biological sample (e.g., cells, blood, plasma, tissue, etc.) from a patient suspected of having cancer, contacting the sample with a labelled antibody (e.g., radioactively tagged antibody) specific for TADG-14, and detecting the TADG-14 protein using standard immunoassay techniques such as an ELISA. Antibody binding to the biological sample indicates that the sample contains a component which specifically binds to an epitope within TADG-14. [0070]
Likewise, a standard Northern blot assay can be used to ascertain the relative amounts of TADG-14 mRNA in a cell or tissue obtained from a patient suspected of having cancer, in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art. This Northern assay uses a hybridization probe, e.g. a full-length, single stranded radiolabelled TADG-14 cDNA probe having a sequence complementary to SEQ ID NO: 6, or a fragment of that DNA sequence at least 20 (preferably at least 30, more preferably at least 50, and most preferably at least 100) consecutive nucleotides in length. The DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art. [0071]
In another embodiment of the present invention, there is provided a method of inhibiting expression of TADG-14 in a cell, comprising the step of: (a) introducing a vector into a cell, whereupon expression of the vector produces TADG-14 antisense DNA which hybridizes to endogenous TADG-14 mRNA and inhibits expression of TADG-14 in the cell. [0072]
Further embodied by the present invention, there is provided a method of inhibiting a TADG-14 protein in a cell, comprising the step of: (a) introducing an antibody specific for TADG-14 protein or a fragment thereof into a cell, whereupon binding of the antibody to the TADG-14 protein inhibits the TADG-14 protein in said cell. [0073]
The present invention is also directed toward a method of targeted therapy to an individual, comprising the step of: (a) administering a compound having a targeting moiety and a therapeutic moiety to an individual, wherein the targeting moiety is specific for TADG-14. Representative targeting moieties include an antibody specific for TADG-14 and a ligand or ligand binding domain that binds TADG-14. Likewise, a representative therapeutic moiety is a radioisotope, a toxin, a chemotherapeutic agent and immune stimulants. Typically, the above-described method is useful when the individual suffers from ovarian cancer, breast cancer or cancers of the prostate, lung, colon and cervix. [0074]
The present invention also provides a method of diagnosing cancer in an individual, comprising the steps of: (a) obtaining a biological sample from an individual; and (b) detecting TADG-14 in the sample. Generally, the presence of TADG-14 in the sample is indicative of the presence of carcinoma in the individual. Generally, the biological sample is blood, ascites fluid, interstitial fluid, tumor tissue biopsy or tumor cells. Typical means of detecting TADG-14 are by Northern blot, Western blot, PCR, dot blot, ELISA, radioimmunoassay, DNA chips or tumor cell labeling. This method may be useful in diagnosing cancers such as ovarian, breast, prostate and colon cancers. [0075]
In still yet another embodiment of the present invention, there is provided a method of vaccinating an individual against TADG-14 protein, comprising the step of (a) inoculating an individual with a TADG-14 protein or fragment thereof which lacks TADG-14 protease activity. Typically, inoculation with the TADG-14 protein or fragment thereof elicits an immune response in the individual, thereby vaccinating the individual against TADG-14. Generally, the individual has cancer, is suspected of having cancer or is at risk of getting cancer. Preferably, the TADG-14 fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, or 64. [0076]
In another embodiment of the present invention, there is provided a method of producing activated immune cells directed toward TADG-14, comprising the steps of exposing immune cells to a TADG-14 protein or fragment thereof which lacks TADG-14 protease activity. Usually, exposure to the TADG-14 protein or fragment thereof activates the immune cells, thereby producing activated immune cells directed toward TADG-14. Generally, the immune cells are B cells, T cells or dendritic cells. Preferably, the dendritic cells are isolated from an individual prior to exposure to a TADG-14 protein or fragment thereof, and then reintroduced into the individual subsequent to the exposure. Typically, the individual has cancer, is suspected of having cancer or is at risk of getting cancer. Preferably, the TADG-14 fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, or 64. [0077]
In another embodiment of the present invention, there is provided an immunogenic composition, comprising an immunogenic fragment of a TADG-14 protein and an appropriate adjuvant. Preferably, the TADG-14 fragment is a 9-residue fragment up to a 20-residue fragment, and more preferably, the 9-residue fragment is SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, or 64. [0078]
In another embodiment of the present invention, there is provided an oligonucleotide having a sequence complementary to SEQ ID No. 6, as well as a composition comprising the oligonucleotide and a physiologically acceptable carrier. Additionally, there is provided a method of treating a neoplastic state in an individual in need of such treatment, comprising the step of (a) administering to the individual an effective dose of the above- described oligonucleotide. [0079]
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. [0080]

EXAMPLE 1

Cloning and characterization of TAD -14 [0081]
TISSUE COLLECTION AND STORAGE: Upon patient hysterectomy, bilateral salpingo-oophorectomy, or surgical removal of neoplastic tissue, the specimen was retrieved and placed it on ice. [0082]
The specimen was then taken to the resident pathologist for isolation and identification of specific tissue samples. Finally, the sample was frozen in liquid nitrogen, logged into the laboratory record and stored at −80° C. Additional specimens were frequently obtained from the Cooperative Human Tissue Network (CHTN). These samples were prepared by the Cooperative Human Tissue Network and shipped on dry ice. Upon arrival, these specimens were logged into the laboratory record and stored at −80° C. [0083]
mRNA ISOLATION AND CDNA SYNTHESIS: Messenger RNA (mRNA) isolation was performed according to the manufacturer's instructions using the Mini RiboSep™ Ultra mRNA isolation kit purchased from Becton Dickinson. This was an oligo(dT) chromatography based system of mRNA isolation. The amount of mRNA recovered was quantitated by UV spectrophotometry. [0084]
First strand complementary DNA (cDNA) was synthesized using 5.0 ug of mRNA and either random hexamer or oligo(dT) primers according to the manufacturer's protocol utilizing a first strand synthesis kit obtained from Clontech. The purity of the cDNA was evaluated by PCR using primers specific for the p53 gene. These primers span an intron such that pure cDNA can be distinguished from cDNA that is contaminated with genomic DNA. [0085]
PCR REACTIONS: Reactions with degenerate primers and quantitative PCR reactions were carried out as previously described (10,11). The sequences of the TADG-14 specific primers that produce the 230 bp product were as follows: 5′-ACAGTACGCCTGGGAGACCA-3′ (SEQ ID No. 12) and 5′-CTGAGACGGTGCAATTCTGG-3′ (SEQ ID No. 13). [0086]
T-VECTOR LIGATION AND TRANSFORMATIONS. The purified PCR products were ligated into the Promega T-vector plasmid and the ligation products were used to transform JM 109 competent cells according to the manufacturer's instructions. Positive colonies were cultured for amplification, the plasmid DNA isolated by means of the Wizard™ Minipreps DNA purification system, and the plasmids were digested with Apal and SacI restriction enzymes to determine the size of the insert. Plasmids with inserts of the size(s) visualized by the previously described PCR product gel electrophoresis were sequenced. [0087]
DNA SEQUENCING: Utilizing a plasmid specific primer near the cloning site, sequencing reactions were carried out using PRISM™ Ready Reaction Dye Deoxy™ terminators (Applied Biosystems) according to the manufacturer's instructions. Residual dye terminators were removed from the completed sequencing reaction using a Centri-sep™ spin column (Princeton Separation). An Applied Biosystems Model 373A DNA Sequencing System was used for sequence analysis. Sequences were compared to GenEMBL databases using the FASTA program ([0088] Wisconsin Package Version 9. 1, Genetics Computer Group (GCG), Madison, Wis.) Multiple sequence alignments were generated with the Bestfit and Pileup programs available through Genetics Computer Group.
NORTHERN BLOT ANALYSIS: mRNAs (approximately 5 μg) were size separated by electrophoresis through a 6.3 % formaldehyde, 1.2% agarose gel in 0.02 M MOPS, 0.05 M sodium acetate (pH 7.0), and 0.001 M EDTA. The mRNAs were then blotted to Hybond—N (Amersham) by capillary action in 20×SSPE. The RNAs were fixed to the membrane by baking for 2 hours at 80° C. Additional multiple tissue northern (MTN) blots were purchased from CLONTECH Laboratories, Inc., including the Human multiple tissue northern blot (cat.#7760-1), the Human multiple tissue northern II blot (cat.#7759-1), the Human Fetal multiple tissue northern II blot (cat.#7756-1), and the Human Brain multiple tissue northern III blot (cat.#7750-1). The 230 bp TADG-14 specific PCR product was radiolabelled utilizing the Prime-a-Gene Labelling System available from Promega. The blots were probed and stripped according to the ExpressHyb Hybridization Solution protocol available from CLONTECH. [0089]
ANTIBODY PRODUCTION AND WESTERN BLOT ANALYSIS Polyclonal antibodies were generated by immunization of white New Zealand rabbits with one of three poly-lysine linked multiple antigen peptides derived from the deduced amino acid sequence of TADG-14. These sequences are KYTVRLGDHSLQ (T14-1, SEQ ID No. 14), GHECQPHSQPWQ (T14-2, SEQ ID No. 15), and LDWIKKIIGSKG (T14-3, SEQ ID No. 16). For Western blot analysis, approximately 20 ug of MDA-MB-435S and HeLa cell lysates were separated on a 15% SDS- PAGE gel and electroblotted to PVDF at 100V for 40 minutes at 4° C. The blot was blocked overnight in Tris-buffered saline (TBS), pH 7.8 containing 0.2% non-fat milk. Primary antibody was added to the membrane at a dilution of 1:100 in 0.2% milk/TBS and incubated for 2 hours at room temperature. The blot was washed and incubated with a 1:3000 dilution of alkaline-phosphatase conjugated goat anti-rabbit IgG antibody (Bio-Rad) for one hour at room temperature. The blot was washed and incubated with a chemiluminescent substrate (Bio-Rad) before a 10-second exposure to X-ray film for visualization. [0090]
IMMUNOHISTOCHEMISTRY: Immunohistochemical staining was performed using a Vectastain Elite ABC Kit (Vector). Formalin fixed and paraffin embedded specimens were routinely deparaffinized and processed using microwave heat treatment in 0.01 M sodium citrate buffer (pH 6.0). The specimens were incubated in methanol with 0.3% H[0091] ₂O₂for 30 minutes at room temperature and then incubated with normal goat serum for 30 minutes. The samples were incubated with anti-TADG-14 peptide derived polyclonal antibody for 1 hour at room temperature in a moisture chamber, followed by incubation with biotinylated anti- rabbit IgG for 30 minutes, and then incubated with ABC reagent (Vector) for 30 minutes. The final products were visualized using the AEC substrate system (DAKO) and sections were counter stained with hematoxylin before mounting. Negative controls were performed by using normal serum instead of the primary antibody.
Results [0092]
The gene encoding the novel extracellular serine protease of the present invention was identified from a group of proteases overexpressed in carcinoma by subcloning and sequencing the appropriate PCR products. An example of such a PCR reaction is given in FIG. 1. Subcloning and sequencing of individual bands from such an amplification provided a basis for identifying the novel protease of the present invention. [0093]
After confirming the 406 bp PCR product was unique and was appropriately conserved to fit into the serine protease family, this PCR product was used as a probe for Northern blot analysis to determine the transcript size and tissue specificity of its expression. It was found that the mRNA for this clone is approximately 1.4 kilobases (kb) (FIG. 2A), and that it is strongly expressed in ovarian carcinomas but not in normal ovary. More importantly, the transcript was found to be undetectable by Northern analysis in 28 normal human tissues studied (FIG. 2B, C, D and data not shown). In a more sensitive assay of 50 normal human tissues (Clontech), RNA dot blot analysis revealed that this clone was very weakly expressed in only three of these 50 tissues, kidney, lung and mammary gland (data not shown). [0094]
Using standard hybridization techniques, a cDNA library constructed from the mRNA isolated from the ascites cells of an ovarian cystadenocarcinoma patient was screened. Five clones were obtained, two of which overlapped and spanned 1343 nucleotides (FIG. 3A). The last two nucleotides prior to the poly (A) tail and the poly (A) tail itself were obtained from the EST database available a t NCBI (accession #AA343629). Subsequent Northern blot analyses with probes derived from sequences near the 5′ or 3′ end of this cDNA were consistent with previous results suggesting that the obtained clones were produced by the same gene (data not shown). This cDNA includes a Kozak's consensus sequence for the initiation of translation, and a polyadenylation signal. [0095]
The mRNA provides an open reading frame of 260 amino acids, which contains the necessary residues (His[0096] ⁷³, Asp¹²⁰, Ser²¹²) in the appropriate context to classify this protein as a trypsin-like serine protease (1). Near its amino-terminus, the predicted protein contains a stretch of hydrophobic amino acids that probably serve as a secretion signal sequence. In addition, residues 110 to 112 encode a potential site for glycosylation that is common to serine proteases of the kallikrein subfamily such as PSA. This enzyme was named TADG-14, and the sequence was submitted to GenBank and assigned the accession # AF055982.
Comparison of the deduced TADG-14 amino acid sequence with sequences of known proteases revealed that i t possesses significant similarity with human glandular kallikrein (hHk2), PSA, Protease M and mouse neuropsin (11-14). The sequence determined for the catalytic domain of TADG-14 is presented in FIG. 4 and is consistent with other serine proteases and specifically contains conserved amino acids appropriate for the catalytic domain of the serine protease family. At the amino acid level TADG-14 is 48% identical to Protease M, 46% identical to hHk2, and 43% identical to PSA (FIG. 3B). More interestingly, the mouse protease neuropsin and TADG-14 share 72% amino acid identity. In addition to the similarity of the protein sequences, neuropsin and TADG-14 mRNAs are of similar size (1.4 kb) and structure with approximately the same amounts of 5′ and 3′ untranslated regions suggesting the possibility of orthology. Neuropsin was originally identified as being expressed in mouse hippocampus and shown to be differentially expressed under stimulation (14). However, TADG-14 mRNA was undetectable in human whole brain by Northern blot. Further, Northern blot analysis for TADG-14 in eight separate parts of human brain including amygdala, caudate nucleus, corpus callosum, hippocampus, whole brain, substantia nigra, subthalamic nucleus and thalmus, also turned out to be negative. Recently, a human cDNA encoding neuropsin has been submitted to the GenBank database (accession # AB009849). Although this clone represents a different transcript from TADG-14, it encodes a protein that is identical to TADG-14 (15). Therefore, it seems logical that TADG-14 and neuropsin may arise as alternative splicing products from the same gene. [0097]
To characterize the extent and frequency of expression of the TADG-14 gene in ovarian tumors, semi-quantitative PCR was performed with cDNA derived from normal ovary, ovarian carcinoma or low malignant potential (LMP) tumors as template. This technique has been previously authenticated and verified by Northern blot, Western blot and immunohistochemistry (9, 16). PCR primers that amplify a TADG-14 specific 230 bp product were synthesized and used simultaneously in reactions with primers that produce a specific 454 bp PCR product for β-tubulin. A radiolabelled nucleotide was included in this reaction, the PCR products were separated on a 2% agarose gel and the intensity of each band was quantitated by a Phosphoimager (Molecular Dynamics). FIG. 5A shows an ethidium bromide stained agarose gel with the separated quantitative PCR products and is representative of the typical results observed. [0098]

The ratio of the TADG-14 PCR product to that of β-tubulin (mean±SD) was calculated for normal ovary samples which all showed relatively low expression levels (0.034±0.024). TADG-14 overexpression was defined as exceeding the mean of the ratio of TADG-14 to β-tubulin for normal samples by greater than 2 standard deviations (SD). TADG-14 was found to be overexpressed in 4 of 10 LMP tumors (40%), and 20 of 30 ovarian carcinomas (67%) studied. For individual histologic subtypes of tumor, the expression ratio was 0.110±0.092 for serous LMP tumors, 0.096±0.142 for mucinous LMP tumors, 0.457±0.345 for serous carcinomas, 0.171±0.300 for mucinous carcinomas, 0.308±0.144 for clear cell carcinomas, and 0.485±0.325 for endometrioid carcinomas. Of the 30 ovarian carcinomas studied, 13 of 17 serous tumors, 1 of 7 mucinous tumors, 3 of 3 clear cell tumors and 3 of 3 endometrioid tumors overexpressed TADG-14 (FIG. 5B). These data are summarized in Table 1. Although not quantitated, transcripts for TADG-14 were also detectable in breast and colon carcinoma (FIG. 6).

TABLE 1


TADG-14 Overexpression by Tissue Subtype

		TADG14
	Tissue Type	Overexpression

Normal

0/10	(0%)
LMP	4/10	(40%)
Serous	3/6	(50%)
Mucinous	1/4	(25%)
Carcinoma	20/30	(67%)
Serous	13/17	(76%)
Mucinous	1/7	(14%)
Endometrioid	3/3	(100%)
Clear Cell	3/3	(100%)

Immunogenic poly-lysine linked multiple antigen peptides were synthesized based on the deduced amino acid sequence of TADG-14 and used to immunize rabbits for the production of polyclonal antibodies. The antiserum raised to the peptide sequence LDWIKKIIGSKG (SEQ ID No. 16), near the carboxy terminal (AA #249-260), was used in Western blot analysis to determine if this antibody would recognize a protein of the predicted size of 28 kDa. Proteins from the HeLa cell line and the carcinoma derived MD-MBA-435S cell line were used in this experiment and it was found that the antibody recognized a single 30 kDa protein in both cell lines (FIG. 7, [0100] lanes 3 and 4). This size is within a reasonable range of the predicted molecular weight. As a negative control, duplicate HeLa and MD-MB435S lysates were examined with rabbit pre-immune serum (FIG. 7, lanes 1 and 2). More importantly, this experiment was reproducible with antisera to a peptide from a different region of TADG-14, suggesting that these cultured cancer cells produce the TADG-14 protein.
Immunohistochemical staining supported the data obtained by quantitative PCR and Northern blot. Using a TADG-14 peptide directed antibody, no staining was observed with normal ovarian tissue samples (FIG. 8A). However, intense staining was associated with tumor cells of all of the various histological subtypes of ovarian carcinoma examined. For serous carcinoma (FIG. 8B and 8C) the antigen appears to be associated with tumor cells in the form of granules. These granular structures may be intermediates in the pathway that ultimately leads to secretion of TADG-14 In mucinous and clear cell carcinoma samples (FIGS. 8D and F respectively), TADG-14 is highly associated with the tumor cells. In endometrioid carcinoma (FIG. 8E), the antigen is most prevalent in the glandular lumen formed by the tumor cells. [0101]
The lethality of neoplastic cells lies in their ability to proliferate abnormally and invade normal host tissues. Malignancies employ proteases to provide a variety of services that assist in the process of tumor progression including activation of growth and angiogenic factors and to provide the basis for invasion and metastasis. In the process of studying these enzymes, overexpression of known proteases such as hepsin and SCCE have been identified. In the present study, a cDNA encoding a novel serine protease, TADG14, was cloned. This protease was found to be very highly expressed in 67% (20/30) of ovarian carcinomas studied, whereas it was undetected in normal ovarian tissue. The TADG14 transcript was also not detectable in any of 50 normal human tissues studied. On prolonged Northern blot exposure, extremely low levels of TADG-14 were detected in normal kidney, breast and lung. This suggests the possibility that this gene is under the control of a promoter that is most active in ovarian tumors, and it may be possible to exploit this for therapeutic means. Unfortunately, TADG14 expression can be detected in other types of cancer including prostate, breast and colon. This may limit the usefulness of TADG14 as a potential diagnostic maker for ovarian carcinoma, but it in no way detracts from the usefulness of this molecule as a target for cancer therapy or the usefulness of the TADG14 promoter in gene therapy applications. [0102]
At the nucleotide level, TADG14 mRNA resembles the recently cloned human neuropsin transcript with obvious differences residing in the 5′ and 3′ UTRS. TADG14 mRNA contains 491 bases of 5′ UTR that were not found in human neuropsin. Also, the nucleotides preceding the poly (A) tail in the 3′ UTR are not homologous. A O.9 kb transcript for human neuropsin was identified in cultured keratinocytes but not in normal hippocampus. Also, it was not identified as being associated with tumors. At the amino acid level, TADG14 is identical to human neuropsin. [0103]
Among other known proteases, TADG14 most closely resembles the mouse protease known as neuropsin, which was originally cloned from mouse hippocampus, and subsequently implicated in neuronal plasticity (17). If TADG14 functions in a manner similar to mouse neuropsin, it may be capable of restructuring the three-dimensional architecture of a tumor allowing for shedding of tumor cells or invasion of normal host tissues by degrading fibronectin (18). In support of this, immunohistochemical staining of ovarian tumors revealed that TADG14 is highly associated with tumor cells and the cells near the invasive fronts of tumor. Therefore, TADG14 could be an important target for the inhibition of tumor progression. [0104]
Most importantly, the five-year survival rate for ovarian cancer patients remains below 50% because of an inability to diagnose this disease at an early stage. TADG14 contains a secretion signal sequence and immunohistochemical data suggest that TADG14 is secreted. In addition, by Northern blot and RNA dot blot analyses, TADG14 appears only in abundance in tumor tissues. As a result of this, it may be possible to design assays based on the detection of this protein for the early detection of ovarian cancer. Currently, the best available ovarian cancer tumor marker is CA125. However, due to high endogenous circulating levels of this antigen, the signal to noise ratio limits its usefulness as a diagnostic tool. Therefore, TADG14, due to its limited expression in other tissues and potential for being present in the circulation of tumor bearing patients, may prove to be a useful tool for early detection of ovarian cancer, especially the most prevalent serous cystadenocarcinoma subtype. [0105]

EXAMPLE 2

Peptide ranking analysis for vaccine candidates [0106]

For vaccine or immune stimulation, individual 9-mers to 11-mers of the TADG-14 protein were examined to rank the binding of individual peptides to the top 8 haplotypes in the general population (Parker et al., (1994)). Table 2 shows the peptide ranking based upon the predicted half-life of each peptidep's binding to a particular HLA allele. A larger half-life indicates a stronger association with that peptide and the particular HLA molecule. The TADG-14 peptides that strongly bind to an HLA allele are putative immunogens, and are used to innoculate an individual against TADG-14.

TABLE 2


TADG-14 peptide ranking

HLA Type
& Ranking			Predicted	SEQ
No.	Start	Peptide	Dissociation_½	ID

HLA A0201
1	55	QLLCGGVLV	257.342	17
2	15	LLLLGGAWA	171.868	18
3	60	GVLVGGNWV	123.846	19
4	61	VLVGGNWVL	111.672	20
5	49	ALFQGQQLL	79.041	21
6	10	KTWMFLLLL	75.331	22
7	131	SLGSKVKPI	23.995	23
8	122	MLLQLRDQA	15.312	24
9	124	LQLRDQASL	13.624	25
10	170	TLNCAEVKI	10.433	26
HLA A0205
1	124	LQLRDQASL	28.560	25
2	10	KTWMFLLLL	25.200	22
3	49	ALFQGQQLL	21.000	21
4	208	CQGDSGGPL	16.800	27
5	54	QQLLCGGVL	16.800	28
6	61	VLVGGNWVL	14.280	20
7	60	GVLVGGNWV	12.000	19
8	62	LVGGNWVLT	6.800	29
9	55	QLLCGGVLV	6.000	17
10	191	QITDGMVCA	3.000	30
HLA A1
1	173	CAEVKTFPQ	4.500	31
2	83	LGDHSLQNK	2.500	32
3	183	KCEDAYPGQ	1.800	33
4	192	ITDGMVCAG	1.250	34
5	71	AAHCKKPKY	1.000	35
6	113	DVEDHNHDL	0.900	36
7	229	GSDPCGRSD	0.750	37
8	111	SSDVEDHNH	0.750	38
9	28	AQEDKVLGG	0.675	39
10	217	VCDGALQGI	0.500	40
HLA A24
1	241	VYTNICRYL	280.000	41
2	247	RYLDWTKKI	198.000	42
3	10	KTWMFLLLL	8.000	22
4	7	RAAKTWMFL	8.000	43
5	42	HSQPWQAAL	7.200	44
6	48	AALFQGQQL	7.200	45
7	113	DVEDHNHDL	7.200	36
8	54	QQLLCGGVL	6.000	28
9	214	GPLVCDGAL	6.000	46
10	61	VLVGGNWVL	6.000	20
HLA B7
1	80	TVRLGDHSL	200.000	47
2	5	RPRAAKTWM	200.000	48
3	214	GPLVCDGAL	80.000	46
4	48	AALFQGQQL	36.000	45
5	8	AAKTWMFLL	36.000	49
6	3	RPRPPAAKT	20.000	50
7	162	SPRENFPDT	20.000	51
8	188	YPGQITDGM	20.000	52
9	7	RAAKTWMFL	12.000	43
10	49	ALFQGQQLL	12.000	21
HLA B8
1	133	GSKVKPISL	80.000	53
2	8	AAKTWMFLL	16.000	49
3	3	RPRPRAAKT	8.000	50
4	80	TVRLGDHSL	4.000	47
5	73	HCKKPKYTV	2.400	54
6	131	SLGSKVKPI	2.000	23
7	5	RPRAAKTWM	2.000	48
8	162	SPRENFPDT	1.200	51
9	214	GPLVCDGAL	0.800	46
10	179	FPQKKCEDA	0.800	55
HLA B2702
1	234	GRSDKPGVY	200.000	56
2	246	CRYLDWIKK	20.000	57
3	101	VQSIPHPCY	20.000	58
4	43	SQPWQAALF	20.000	59
5	6	PRAAKTWMF	20.000	60
6	26	SRAQEDKVL	18.000	61
7	126	LRDQASLGS	10.000	62
8	149	GQKCTVSGW	10.000	63
9	124	LQLRDQASL	6.000	25
10	54	QQLLCGGVL	6.000	28
HLA B4403
1	96	QEIPVVQSI	180.000	64
2	71	AAHCKKPKY	13.500	35
3	171	LNCAEVKIF	4.500	65
4	184	CEDAYPGQI	4.000	66
5	114	VEDHNHDLM	4.000	67
6	101	VQSIPHPCY	2.250	68
7	236	SDKPGVYTN	1.800	69
8	164	RENFPDTLN	1.800	70
9	174	AEVKIFPQK	1.600	71
10	43	SQPWQAALF	1.500	59

The following references were cited herein: [0108]
1. Neurath, H. The Diversity of Proteolytic Enzymes. In: R. J. Beynon and J. S. Bond (eds.), pp. 1-13, Proteolytic enzymes, Oxford: IRL Press, 1989 [0109]
2. Liotta et al., Cell, 64: 327-336, 1991. [0110]
3. Duffy, Clin. Exp. Metastasis, 10: 145-155, 1992. [0111]
4. Tryggvason et al., Biochem. Biophys. Acta., 907: 191-217, 1987. [0112]
5. Monsky et al., Cancer Res., 53: 3159-3164, 1993. [0113]
6. Powell et al., Cancer Res., 53: 417-422, 1993. [0114]
7. McCormack et al., Urology, 45: 729-744, 1995. [0115]
8. Sakanari et al., Proc. Natl. Acad. Sci. U.S.A., 86: 4863-4867, 1989. [0116]
9. Tanimoto et al., Cancer Res., 57: 2884-2887, 1997. [0117]
10. Tanimoto et al., Cancer, 1998. [0118]
11. Evans et al., Biochemistry, 27: 3124-3129, 1988. [0119]
12. Watt et al., Proc. Natl. Acad. Sci. U.S.A., 83: 3166-3120, 1986. [0120]
13. Anisowicz et al., Mol. Medicine. 2: 624-636, 1996. [0121]
14. Chen et al., J. Neuroscience. 15: 5088-5097, 1995. [0122]
15. Yoshida et al., Gene 213: 9-16, 1998. [0123]
16. Shigemasa et al., J. Soc. Gynecol. Invest. 4: 95-102, 1997. [0124]
17. Suzuki et al., Neuroscience Res., 23: 345-351, 1995. [0125]
18. Shimizu et al., J Biol Chem, 273: 11189-11196, 1998. [0126]
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. [0127]
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims. [0128]

0

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 72

<210> SEQ ID NO 1

<211> LENGTH: 144

<212> TYPE: PRT

<213> ORGANISM: unknown

<220> FEATURE:

<221> NAME/KEY: DOMAIN

<223> OTHER INFORMATION: Amino acid sequence of Protease m (Prom)

catalytic domain

<400> SEQUENCE: 1

Trp Val Leu Thr Ala Ala His Cys Lys Lys Pro Asn Leu Gln Val

5 10 15

Phe Leu Glu Lys His Asn Leu Arg Gln Arg Glu Ser Ser Gln Glu

20 25 30

Gln Ser Ser Val Val Arg Ala Val Ile His Pro Asp Tyr Asp Ala

35 40 45

Ala Ser His Asp Gln Asp Ile Met Leu Leu Arg Leu Ala Arg Pro

50 55 60

Ala Lys Leu Ser Glu Leu Ile Gln Pro Leu Pro Leu Glu Arg Asp

65 70 75

Cys Ser Ala Asn Thr Thr Ser Cys His Ile Leu Gly Trp Gly Lys

80 85 90

Thr Ala Asp Gly Asp Phe Pro Asp Thr Ile Gln Cys Ala Tyr Ile

95 100 105

His Leu Val Ser Arg Glu Glu Cys Glu His Ala Tyr Pro Gly Gln

110 115 120

Ile Thr Gln Asn Met Leu Cys Ala Gln Asp Glu Lys Tyr Gly Lys

125 130 135

Asp Ser Cys Gln Gly Asp Ser Gly Gly

140

<210> SEQ ID NO 2

<211> LENGTH: 148

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<222> LOCATION: DOMAIN

<223> OTHER INFORMATION: Amino acid sequence of Tadg14 catalytic domain

<400> SEQUENCE: 2

Trp Val Val Thr Ala Ala His Cys Lys Lys Pro Lys Tyr Thr Val

5 10 15

Arg Leu Gly Asp His Ser Leu Gln Asn Lys Asp Gly Pro Glu Gln

20 25 30

Glu Ile Pro Val Val Gln Ser Ile Pro His Pro Cys Tyr Asn Ser

35 40 45

Ser Asp Val Glu Asp His Asn His Asp Leu Met Leu Leu Gln Leu

50 55 60

Arg Asp Gln Ala Ser Leu Gly Ser Lys Val Lys Pro Ile Ser Leu

65 70 75

Ala Asp His Cys Thr Gln Pro Gly Gln Asn Cys Thr Val Ser Gly

80 85 90

Trp Gly Thr Val Thr Ser Pro Arg Glu Asn Phe Pro Asp Thr Leu

95 100 105

Asn Cys Ala Glu Val Lys Ile Phe Pro Gln Lys Lys Cys Glu Asp

110 115 120

Ala Tyr Pro Gly Gln Ile Thr Asp Gly Met Val Cys Ala Gly Ser

125 130 135

Ser Lys Gly Ala Asp Thr Cys Gln Gly Asp Ser Gly Gly

140 145

<210> SEQ ID NO 3

<211> LENGTH: 146

<212> TYPE: PRT

<213> ORGANISM: unknown

<220> FEATURE:

<221> NAME/KEY: DOMAIN

<223> OTHER INFORMATION: Amino acid sequence of trypsin like serine

protease (Try1) catalytic domain

<400> SEQUENCE: 3

Trp Val Val Ser Ala Gly His Cys Tyr Lys Ser Arg Ile Gln Val

5 10 15

Arg Leu Gly Glu His Asn Ile Glu Val Leu Glu Gly Asn Glu Gln

20 25 30

Phe Ile Asn Ala Ala Lys Ile Ile Arg His Pro Gln Tyr Asp Arg

35 40 45

Lys Thr Leu Asn Asn Asp Ile Met Leu Ile Lys Leu Ser Ser Arg

50 55 60

Ala Val Ile Asn Ala Arg Val Ser Thr Ile Ser Leu Pro Thr Ala

65 70 75

Pro Pro Ala Thr Gly Thr Lys Cys Leu Ile Ser Gly Trp Gly Asn

80 85 90

Thr Ala Ser Ser Gly Ala Asp Tyr Pro Asp Glu Leu Gln Cys Leu

95 100 105

Asp Ala Pro Val Leu Ser Gln Ala Lys Cys Glu Ala Ser Tyr Pro

110 115 120

Gly Lys Ile Thr Ser Asn Met Phe Cys Val Gly Phe Leu Glu Gly

125 130 135

Gly Lys Asp Ser Cys Gln Gly Asp Ser Gly Gly

140 145

<210> SEQ ID NO 4

<211> LENGTH: 144

<212> TYPE: PRT

<213> ORGANISM: unknown

<220> FEATURE:

<221> NAME/KEY: DOMAIN

<223> OTHER INFORMATION: Amino acid sequence of stratum corneum

chymotryptic enzyme (scce) catalytic domain

<400> SEQUENCE: 4

Trp Val Leu Thr Ala Ala His Cys Lys Met Asn Glu Tyr Thr Val

5 10 15

His Leu Gly Ser Asp Thr Leu Gly Asp Arg Arg Ala Gln Arg Ile

20 25 30

Lys Ala Ser Lys Ser Phe Arg His Pro Gly Tyr Ser Thr Gln Thr

35 40 45

His Val Asn Asp Leu Met Leu Val Lys Leu Asn Ser Gln Ala Arg

50 55 60

Leu Ser Ser Met Val Lys Lys Val Arg Leu Pro Ser Arg Cys Glu

65 70 75

Pro Pro Gly Thr Thr Cys Thr Val Ser Gly Trp Gly Thr Thr Thr

80 85 90

Ser Pro Asp Val Thr Phe Pro Ser Asp Leu Met Cys Val Asp Val

95 100 105

Lys Leu Ile Ser Pro Gln Asp Cys Thr Lys Val Tyr Lys Asp Leu

110 115 120

Leu Glu Asn Ser Met Leu Cys Ala Gly Ile Pro Asp Ser Lys Lys

125 130 135

Asn Ala Cys Asn Gly Asp Ser Gly Gly

140

<210> SEQ ID NO 5

<211> LENGTH: 159

<212> TYPE: PRT

<213> ORGANISM: unknown

<220> FEATURE:

<221> NAME/KEY: DOMAIN

<223> OTHER INFORMATION: Amino acid sequence of hepsin (heps) catalytic

domain

<400> SEQUENCE: 5

Trp Val Leu Thr Ala Ala His Cys Phe Pro Glu Arg Asn Arg Val

5 10 15

Leu Ser Arg Trp Arg Val Phe Ala Gly Ala Val Ala Gln Ala Ser

20 25 30

Pro His Gly Leu Gln Leu Gly Val Gln Ala Val Val Tyr His Gly

35 40 45

Gly Tyr Leu Pro Phe Arg Asp Pro Asn Ser Glu Glu Asn Ser Asn

50 55 60

Asp Ile Ala Leu Val His Leu Ser Ser Pro Leu Pro Leu Thr Glu

65 70 75

Tyr Ile Gln Pro Val Cys Leu Pro Ala Ala Gly Gln Ala Leu Val

80 85 90

Asp Gly Lys Ile Cys Thr Val Thr Gly Trp Gly Asn Thr Gln Tyr

95 100 105

Tyr Gly Gln Gln Ala Gly Val Leu Gln Glu Ala Arg Val Pro Ile

110 115 120

Ile Ser Asn Asp Val Cys Asn Gly Ala Asp Phe Tyr Gly Asn Gln

125 130 135

Ile Lys Pro Lys Met Phe Cys Ala Gly Tyr Pro Glu Gly Gly Ile

140 145 150

Asp Ala Cys Gln Gly Asp Ser Gly Gly

155

<210> SEQ ID NO 6

<211> LENGTH: 1360

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Nucleotide sequence encoding Tumor Antigen

Derived Gene-14 (TADG-14) protein; nt 1344-1360 NCBI accession

#AA343629

<400> SEQUENCE: 6

ctgtagcagg cagagcttac caagtctctc cgaactcaaa tggaagaaat accttatgaa 60

tgtaagaatg tagggggtca tggcttgtaa tttacacagt gtaaatgaaa ccatcctaga 120

ggattatgag gaatcctttc tatgtgattt tcaatcatag caagcaagaa aggctccagt 180

gtcaaggtag ttcagctctt acaggatata aaacagtcca tacttgagag aaaaaactta 240

gatctgagtg atggaatgtg aagcaaatct ttcaaaatca gtagacattt cttggacata 300

aaacacagat gaggaaaggg cttcaaatta gaagttacgt aatcaccatc agaaagttca 360

tgtttggtaa attctgttac tagaaatgta ggaaattcag gtatagcttt gaatcccaat 420

tacacattgg tcagtgggaa aactaagggc ctccaacagg caaattcagg gaggataggt 480

ttcagggaat gccctggatt ctggaagacc tcaccatggg acgcccccga cctcgtgcgg 540

ccaagacgtg gatgttcctg ctcttgctgg ggggagcctg ggcaggacac tccagggcac 600

aggaggacaa ggtgctgggg ggtcatgagt gccaacccca ttcgcagcct tggcaggcgg 660

ccttgttcca gggccagcaa ctactctgtg gcggtgtcct tgtaggtggc aactgggtcc 720

ttacagctgc ccactgtaaa aaaccgaaat acacagtacg cctgggagac cacagcctac 780

agaataaaga tggcccagag caagaaatac ctgtggttca gtccatccca cacccctgct 840

acaacagcag cgatgtggag gaccacaacc atgatctgat gcttcttcaa ctgcgtgacc 900

aggcatccct ggggtccaaa gtgaagccca tcagcctggc agatcattgc acccagcctg 960

gccagaagtg caccgtctca ggctggggca ctgtcaccag tccccgagag aattttcctg 1020

acactctcaa ctgtgcagaa gtaaaaatct ttccccagaa gaagtgtgag gatgcttacc 1080

cggggcagat cacagatggc atggtctgtg caggcagcag caaaggggct gacacgtgcc 1140

agggcgattc tggaggcccc ctggtgtgtg atggtgcact ccagggcatc acatcctggg 1200

gctcagaccc ctgtgggagg tccgacaaac ctggcgtcta taccaacatc tgccgctacc 1260

tggactggat caagaagatc ataggcagca agggctgatt ctaggataag cactagatct 1320

cccttaataa actcacaact ctctgaaaaa aaaaaaaaaa 1360

<210> SEQ ID NO 7

<211> LENGTH: 260

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of TADG-14 protein

<400> SEQUENCE: 7

Met Gly Arg Pro Arg Pro Arg Ala Ala Lys Thr Trp Met Phe Leu

5 10 15

Leu Leu Leu Gly Gly Ala Trp Ala Gly His Ser Arg Ala Gln Glu

20 25 30

Asp Lys Val Leu Gly Gly His Glu Cys Gln Pro His Ser Gln Pro

35 40 45

Trp Gln Ala Ala Leu Phe Gln Gly Gln Gln Leu Leu Cys Gly Gly

50 55 60

Val Leu Val Gly Gly Asn Trp Val Leu Thr Ala Ala His Cys Lys

65 70 75

Lys Pro Lys Tyr Thr Val Arg Leu Gly Asp His Ser Leu Gln Asn

80 85 90

Lys Asp Gly Pro Glu Gln Glu Ile Pro Val Val Gln Ser Ile Pro

95 100 105

His Pro Cys Tyr Asn Ser Ser Asp Val Glu Asp His Asn His Asp

110 115 120

Leu Met Leu Leu Gln Leu Arg Asp Gln Ala Ser Leu Gly Ser Lys

125 130 135

Val Lys Pro Ile Ser Leu Ala Asp His Cys Thr Gln Pro Gly Gln

140 145 150

Lys Cys Thr Val Ser Gly Trp Gly Thr Val Thr Ser Pro Arg Glu

155 160 165

Asn Phe Pro Asp Thr Leu Asn Cys Ala Glu Val Lys Ile Phe Pro

170 175 180

Gln Lys Lys Cys Glu Asp Ala Tyr Pro Gly Gln Ile Thr Asp Gly

185 190 195

Met Val Cys Ala Gly Ser Ser Lys Gly Ala Asp Thr Cys Gln Gly

200 205 210

Asp Ser Gly Gly Pro Leu Val Cys Asp Gly Ala Leu Gln Gly Ile

215 220 225

Thr Ser Trp Gly Ser Asp Pro Cys Gly Arg Ser Asp Lys Pro Gly

230 235 240

Val Tyr Thr Asn Ile Cys Arg Tyr Leu Asp Trp Ile Lys Lys Ile

245 250 255

Ile Gly Ser Lys Gly

260

<210> SEQ ID NO 8

<211> LENGTH: 260

<212> TYPE: PRT

<213> ORGANISM: Mus sp.

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of mouse neuropsin

homologous to TADG-14; accession no. D30785

<400> SEQUENCE: 8

Met Gly Arg Pro Pro Pro Cys Ala Ile Gln Pro Trp Ile Leu Leu

5 10 15

Leu Leu Phe Met Gly Ala Trp Ala Gly Leu Thr Arg Ala Gln Gly

20 25 30

Ser Lys Ile Leu Glu Gly Arg Glu Cys Ile Pro His Ser Gln Pro

35 40 45

Trp Gln Ala Ala Leu Phe Gln Gly Glu Arg Leu Ile Cys Gly Gly

50 55 60

Val Leu Val Gly Asp Arg Trp Val Leu Thr Ala Ala His Cys Lys

65 70 75

Lys Gln Lys Tyr Ser Val Arg Leu Gly Asp His Ser Leu Gln Ser

80 85 90

Arg Asp Gln Pro Glu Gln Glu Ile Gln Val Ala Gln Ser Ile Gln

95 100 105

His Pro Cys Tyr Asn Asn Ser Asn Pro Glu Asp His Ser His Asp

110 115 120

Ile Met Leu Ile Arg Leu Gln Asn Ser Ala Asn Leu Gly Asp Lys

125 130 135

Val Lys Pro Val Gln Leu Ala Asn Leu Cys Pro Lys Val Gly Gln

140 145 150

Lys Cys Ile Ile Ser Gly Trp Gly Thr Val Thr Ser Pro Gln Glu

155 160 165

Asn Phe Pro Asn Thr Leu Asn Cys Ala Glu Val Lys Ile Tyr Ser

170 175 180

Gln Asn Lys Cys Glu Arg Ala Tyr Pro Gly Lys Ile Thr Glu Gly

185 190 195

Met Val Cys Ala Gly Ser Ser Asn Gly Ala Asp Thr Cys Gln Gly

200 205 210

Asp Ser Gly Gly Pro Leu Val Cys Asp Gly Met Leu Gln Gly Ile

215 220 225

Thr Ser Trp Gly Ser Asp Pro Cys Gly Lys Pro Glu Lys Pro Gly

230 235 240

Val Tyr Thr Lys Ile Cys Arg Tyr Thr Thr Trp Ile Lys Lys Thr

245 250 255

Met Asp Asn Arg Asp

260

<210> SEQ ID NO 9

<211> LENGTH: 262

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of human glandular

kallikrein (hHk2); accession no. P06870

<400> SEQUENCE: 9

Met Trp Phe Val Leu Cys Leu Ala Leu Ser Leu Gly Gly Thr Gly

5 10 15

Ala Ala Pro Pro Pro Ile Gln Ser Arg Ile Val Gly Gly Trp Glu

20 25 30

Gly Glu Gln His Ser Gln Pro Trp Gln Ala Ala Leu Tyr His Phe

35 40 45

Ser Thr Phe Gln Cys Gly Gly Ile Leu Val His Arg Gln Trp Val

50 55 60

Leu Thr Ala Ala His Cys Ile Ser Asp Asn Tyr Gln Leu Trp Leu

65 70 75

Gly Arg His Asn Leu Phe Asp Asp Glu Asn Thr Ala Gln Phe Val

80 85 90

His Val Ser Glu Ser Phe Pro His Pro Gly Phe Asn Met Ser Leu

95 100 105

Leu Glu Asn His Thr Arg Gln Ala Asp Glu Asp Tyr Ser His Asp

110 115 120

Leu Met Leu Leu Arg Leu Thr Glu Pro Ala Asp Thr Ile Thr Asp

125 130 135

Ala Val Lys Val Val Glu Leu Pro Thr Gln Glu Pro Glu Val Gly

140 145 150

Ser Thr Cys Leu Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Asn

155 160 165

Phe Ser Phe Pro Asp Asp Leu Gln Cys Val Asp Leu Lys Ile Leu

170 175 180

Pro Asn Asp Glu Cys Glu Lys Ala His Val Gln Lys Val Thr Asp

185 190 195

Phe Met Leu Cys Val Gly His Leu Glu Gly Gly Lys Asp Thr Cys

200 205 210

Val Gly Asp Ser Gly Gly Pro Leu Met Cys Asp Gly Val Leu Gln

215 220 225

Gly Val Thr Ser Trp Gly Tyr Val Pro Cys Gly Thr Pro Asn Lys

230 235 240

Pro Ser Val Ala Val Arg Val Leu Ser Tyr Val Lys Trp Ile Glu

245 250 255

Asp Thr Ile Ala Glu Asn Ser

260

<210> SEQ ID NO 10

<211> LENGTH: 261

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of Prostate Specific

Antigen (hPSA); accession no. P07288

<400> SEQUENCE: 10

Met Trp Val Pro Val Val Phe Leu Thr Leu Ser Val Thr Trp Ile

5 10 15

Gly Ala Ala Pro Leu Ile Leu Ser Arg Ile Val Gly Gly Trp Glu

20 25 30

Cys Glu Lys His Ser Gln Pro Trp Gln Val Leu Val Ala Ser Arg

35 40 45

Gly Arg Ala Val Cys Gly Gly Val Leu Val His Pro Gln Trp Val

50 55 60

Leu Thr Ala Ala His Cys Ile Arg Asn Lys Ser Val Ile Leu Leu

65 70 75

Gly Arg His Ser Leu Phe His Pro Glu Asp Thr Gly Gln Val Phe

80 85 90

Gln Val Ser His Ser Phe Pro His Pro Leu Tyr Asp Met Ser Leu

95 100 105

Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp Ser Ser His Asp

110 115 120

Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu Thr Asp Ala

125 130 135

Val Lys Val Met Asp Leu Pro Thr Gln Glu Pro Ala Leu Gly Thr

140 145 150

Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu Phe

155 160 165

Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser

170 175 180

Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe

185 190 195

Met Leu Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser

200 205 210

Gly Asp Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln Gly

215 220 225

Ile Thr Ser Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro

230 235 240

Ser Leu Tyr Thr Lys Val Val His Tyr Arg Lys Trp Ile Lys Asp

245 250 255

Thr Ile Val Ala Asn Pro

260

<210> SEQ ID NO 11

<211> LENGTH: 244

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Amino acid sequence of human protease m

(hProM); accession no. U62801

<400> SEQUENCE: 11

Met Lys Lys Leu Met Val Val Leu Ser Leu Ile Ala Ala Ala Trp

5 10 15

Ala Glu Glu Gln Asn Lys Leu Val His Gly Gly Pro Cys Asp Lys

20 25 30

Thr Ser His Pro Tyr Gln Ala Ala Leu Thr Tyr Ser Gly His Leu

35 40 45

Leu Cys Gly Gly Val Leu Ile His Pro Leu Trp Val Leu Thr Ala

50 55 60

Ala His Cys Lys Lys Pro Asn Leu Gln Val Phe Leu Gly Lys His

65 70 75

Asn Leu Arg Gly Arg Glu Ser Ser Gln Glu Gln Ser Ser Val Val

80 85 90

Arg Ala Val Ile His Pro Asp Tyr Asp Ala Ala Ser His Asp Gln

95 100 105

Asp Ile Met Leu Leu Arg Leu Ala Arg Pro Ala Lys Leu Ser Glu

110 115 120

Leu Ile Gln Pro Leu Pro Leu Glu Arg Asp Cys Ser Ala Asn Thr

125 130 135

Thr Ser Cys His Ile Leu Gly Trp Gly Lys Thr Ala Asp Gly Asp

140 145 150

Phe Pro Asp Thr Ile Gln Cys Ala Tyr Ile His Leu Val Ser Arg

155 160 165

Glu Glu Cys Glu His Ala Tyr Pro Gly Gln Ile Thr Gln Asn Met

170 175 180

Leu Cys Ala Gly Asp Glu Lys Tyr Gly Lys Asp Ser Cys Gln Gly

185 190 195

Asp Ser Gly Gly Pro Leu Val Cys Gly Asp His Ile Arg Gly Leu

200 205 210

Val Ser Trp Gly Asn Ile Pro Cys Gly Ser Lys Glu Lys Pro Gly

215 220 225

Val Tyr Thr Asn Val Cys Arg Tyr Thr Asn Trp Ile Gln Lys Thr

230 235 240

Ile Gln Ala Lys

<210> SEQ ID NO 12

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: artificial sequence

<220> FEATURE:

<221> NAME/KEY: primer

<223> OTHER INFORMATION: Sense primer for TADG14 specific PCR

<400> SEQUENCE: 12

acagtacgcc tgggagacca 20

<210> SEQ ID NO 13

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: artificial sequence

<220> FEATURE:

<221> NAME/KEY: primer

<223> OTHER INFORMATION: Anti-sense primer for TADG14 specific PCR

<400> SEQUENCE: 13

ctgagacggt gcaattctgg 20

<210> SEQ ID NO 14

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Peptide sequence of immunogenic poly-lysine

linked multiple antigen (T14-1) derived from TADG-14 used to

produce polyclonal antibodies

<400> SEQUENCE: 14

Lys Tyr Thr Val Arg Leu Gly Asp His Ser Leu Gln

5 10

<210> SEQ ID NO 15

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Peptide sequence of immunogenic poly-lysine

linked multiple antigen (T14-2) derived from TADG-14 used to

produce polyclonal antibodies

<400> SEQUENCE: 15

Gly His Glu Cys Gln Pro His Ser Gln Pro Trp Gln

5 10

<210> SEQ ID NO 16

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Peptide sequence of immunogenic poly-lysine

linked multiple antigen (T14-3) derived from TADG-14 used to

produce polyclonal antibodies

<400> SEQUENCE: 16

Leu Asp Trp Ile Lys Lys Ile Ile Gly Ser Lys Gly

5 10

<210> SEQ ID NO 17

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 55-63 of the TADG-14 protein

<400> SEQUENCE: 17

Gln Leu Leu Cys Gly Gly Val Leu Val

5

<210> SEQ ID NO 18

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 15-23 of the TADG-14 protein

<400> SEQUENCE: 18

Leu Leu Leu Leu Gly Gly Ala Trp Ala

5

<210> SEQ ID NO 19

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 60-68 of the TADG-14 protein

<400> SEQUENCE: 19

Gly Val Leu Val Gly Gly Asn Trp Val

5

<210> SEQ ID NO 20

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 61-69 of the TADG-14 protein

<400> SEQUENCE: 20

Val Leu Val Gly Gly Asn Trp Val Leu

5

<210> SEQ ID NO 21

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 49-57 of the TADG-14 protein

<400> SEQUENCE: 21

Ala Leu Phe Gln Gly Gln Gln Leu Leu

5

<210> SEQ ID NO 22

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 10-18 of the TADG-14 protein

<400> SEQUENCE: 22

Lys Thr Trp Met Phe Leu Leu Leu Leu

5

<210> SEQ ID NO 23

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 131-139 of the TADG-14 protein

<400> SEQUENCE: 23

Ser Leu Gly Ser Lys Val Lys Pro Ile

5

<210> SEQ ID NO 24

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 122-130 of the TADG-14 protein

<400> SEQUENCE: 24

Met Leu Leu Gln Leu Arg Asp Gln Ala

5

<210> SEQ ID NO 25

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 124-132 of the TADG-14 protein

<400> SEQUENCE: 25

Leu Gln Leu Arg Asp Gln Ala Ser Leu

5

<210> SEQ ID NO 26

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 170-178 of the TADG-14 protein

<400> SEQUENCE: 26

Thr Leu Asn Cys Ala Glu Val Lys Ile

5

<210> SEQ ID NO 27

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 208-216of the TADG-14 protein

<400> SEQUENCE: 27

Cys Gln Gly Asp Ser Gly Gly Pro Leu

5

<210> SEQ ID NO 28

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 54-62 of the TADG-14 protein

<400> SEQUENCE: 28

Gln Gln Leu Leu Cys Gly Gly Val Leu

5

<210> SEQ ID NO 29

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 62-70 of the TADG-14 protein

<400> SEQUENCE: 29

Leu Val Gly Gly Asn Trp Val Leu Thr

5

<210> SEQ ID NO 30

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 191-199 of the TADG-14 protein

<400> SEQUENCE: 30

Gln Ile Thr Asp Gly Met Val Cys Ala

5

<210> SEQ ID NO 31

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 173-181 of the TADG-14 protein

<400> SEQUENCE: 31

Cys Ala Glu Val Lys Ile Phe Pro Gln

5

<210> SEQ ID NO 32

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 83-91 of the TADG-14 protein

<400> SEQUENCE: 32

Leu Gly Asp His Ser Leu Gln Asn Lys

5

<210> SEQ ID NO 33

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 183-191 of the TADG-14 protein

<400> SEQUENCE: 33

Lys Cys Glu Asp Ala Tyr Pro Gly Gln

5

<210> SEQ ID NO 34

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 192-200 of the TADG-14 protein

<400> SEQUENCE: 34

Ile Thr Asp Gly Met Val Cys Ala Gly

5

<210> SEQ ID NO 35

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 71-79 of the TADG-14 protein

<400> SEQUENCE: 35

Ala Ala His Cys Lys Lys Pro Lys Tyr

5

<210> SEQ ID NO 36

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 113-121 of the TADG-14 protein

<400> SEQUENCE: 36

Asp Val Glu Asp His Asn His Asp Leu

5

<210> SEQ ID NO 37

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 229-237 of the TADG-14 protein

<400> SEQUENCE: 37

Gly Ser Asp Pro Cys Gly Arg Ser Asp

5

<210> SEQ ID NO 38

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 111-119 of the TADG-14 protein

<400> SEQUENCE: 38

Ser Ser Asp Val Glu Asp His Asn His

5

<210> SEQ ID NO 39

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 28-36 of the TADG-14 protein

<400> SEQUENCE: 39

Ala Gln Glu Asp Lys Val Leu Gly Gly

5

<210> SEQ ID NO 40

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 217-225 of the TADG-14 protein

<400> SEQUENCE: 40

Val Cys Asp Gly Ala Leu Gln Gly Ile

5

<210> SEQ ID NO 41

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 241-249 of the TADG-14 protein

<400> SEQUENCE: 41

Val Tyr Thr Asn Ile Cys Arg Tyr Leu

5

<210> SEQ ID NO 42

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 247-255 of the TADG-14 protein

<400> SEQUENCE: 42

Arg Tyr Leu Asp Trp Ile Lys Lys Ile

5

<210> SEQ ID NO 43

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 7-15 of the TADG-14 protein

<400> SEQUENCE: 43

Arg Ala Ala Lys Thr Trp Met Phe Leu

5

<210> SEQ ID NO 44

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 42-50 of the TADG-14 protein

<400> SEQUENCE: 44

His Ser Gln Pro Trp Gln Ala Ala Leu

5

<210> SEQ ID NO 45

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 48-56 of the TADG-14 protein

<400> SEQUENCE: 45

Ala Ala Leu Phe Gln Gly Gln Gln Leu

5

<210> SEQ ID NO 46

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 214-222 of the TADG-14 protein

<400> SEQUENCE: 46

Gly Pro Leu Val Cys Asp Gly Ala Leu

5

<210> SEQ ID NO 47

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 80-88 of the TADG-14 protein

<400> SEQUENCE: 47

Thr Val Arg Leu Gly Asp His Ser Leu

5

<210> SEQ ID NO 48

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 5-13 of the TADG-14 protein

<400> SEQUENCE: 48

Arg Pro Arg Ala Ala Lys Thr Trp Met

5

<210> SEQ ID NO 49

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 8-16 of the TADG-14 protein

<400> SEQUENCE: 49

Ala Ala Lys Thr Trp Met Phe Leu Leu

5

<210> SEQ ID NO 50

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 3-11 of the TADG-14 protein

<400> SEQUENCE: 50

Arg Pro Arg Pro Arg Ala Ala Lys Thr

5

<210> SEQ ID NO 51

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 162-170 of the TADG-14 protein

<400> SEQUENCE: 51

Ser Pro Arg Glu Asn Phe Pro Asp Thr

5

<210> SEQ ID NO 52

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 188-196 of the TADG-14 protein

<400> SEQUENCE: 52

Tyr Pro Gly Gln Ile Thr Asp Gly Met

5

<210> SEQ ID NO 53

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 133-141 of the TADG-14 protein

<400> SEQUENCE: 53

Gly Ser Lys Val Lys Pro Ile Ser Leu

5

<210> SEQ ID NO 54

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 73-81 of the TADG-14 protein

<400> SEQUENCE: 54

His Cys Lys Lys Pro Lys Tyr Thr Val

5

<210> SEQ ID NO 55

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 179-187 of the TADG-14 protein

<400> SEQUENCE: 55

Phe Pro Gln Lys Lys Cys Glu Asp Ala

5

<210> SEQ ID NO 56

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 234-242 of the TADG-14 protein

<400> SEQUENCE: 56

Gly Arg Ser Asp Lys Pro Gly Val Tyr

5

<210> SEQ ID NO 57

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 246-254 of the TADG-14 protein

<400> SEQUENCE: 57

Cys Arg Tyr Leu Asp Trp Ile Lys Lys

5

<210> SEQ ID NO 58

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 101-109 of the TADG-14 protein

<400> SEQUENCE: 58

Val Gln Ser Ile Pro His Pro Cys Tyr

5

<210> SEQ ID NO 59

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 43-51 of the TADG-14 protein

<400> SEQUENCE: 59

Ser Gln Pro Trp Gln Ala Ala Leu Phe

5

<210> SEQ ID NO 60

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 6-14 of the TADG-14 protein

<400> SEQUENCE: 60

Pro Arg Ala Ala Lys Thr Trp Met Phe

5

<210> SEQ ID NO 61

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 26-34 of the TADG-14 protein

<400> SEQUENCE: 61

Ser Arg Ala Gln Glu Asp Lys Val Leu

5

<210> SEQ ID NO 62

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 126-134 of the TADG-14 protein

<400> SEQUENCE: 62

Leu Arg Asp Gln Ala Ser Leu Gly Ser

5

<210> SEQ ID NO 63

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 149-157 of the TADG-14 protein

<400> SEQUENCE: 63

Gly Gln Lys Cys Thr Val Ser Gly Trp

5

<210> SEQ ID NO 64

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 96-104 of the TADG-14 protein

<400> SEQUENCE: 64

Gln Glu Ile Pro Val Val Gln Ser Ile

5

<210> SEQ ID NO 65

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 171-179 of the TADG-14 protein

<400> SEQUENCE: 65

Leu Asn Cys Ala Glu Val Lys Ile Phe

5

<210> SEQ ID NO 66

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 184-192 of the TADG-14 protein

<400> SEQUENCE: 66

Cys Glu Asp Ala Tyr Pro Gly Gln Ile

5

<210> SEQ ID NO 67

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 114-122 of the TADG-14 protein

<400> SEQUENCE: 67

Val Glu Asp His Asn His Asp Leu Met

5

<210> SEQ ID NO 68

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 101-109 of the TADG-14 protein

<400> SEQUENCE: 68

Val Gln Ser Ile Pro His Pro Cys Tyr

5

<210> SEQ ID NO 69

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 236-244 of the TADG-14 protein

<400> SEQUENCE: 69

Ser Asp Lys Pro Gly Val Tyr Thr Asn

5

<210> SEQ ID NO 70

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 164-172 of the TADG-14 protein

<400> SEQUENCE: 70

Arg Glu Asn Phe Pro Asp Thr Leu Asn

5

<210> SEQ ID NO 71

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Homo sapiens

<220> FEATURE:

<223> OTHER INFORMATION: Residues 174-182 of the TADG-14 protein

<400> SEQUENCE: 71

Ala Glu Val Lys Ile Phe Pro Gln Lys

5

<210> SEQ ID NO 72

<211> LENGTH: 1343

<212> TYPE: DNA

<213> ORGANISM: artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Anti-sense sequence of TADG-14

<400> SEQUENCE: 72

gaattccgtg agtttattaa gggagatcta gtgcttatcc tagaatcagc 50

ccttgctgcc tatgatcttc ttgatccagt ccaggtagcg gcagatgttg 100

gtatagacgc caggtttgtc ggacctccca caggggtctg agccccagga 150

tgtgatgccc tggagtgcac catcacacac cagggggcct ccagaatcgc 200

cctggcacgt gtcagcccct ttgctgctgc ctgcacagac catgccatct 250

gtgatctgcc ccgggtaagc atcctcacac ttcttctggg gaaagatttt 300

tacttctgca cagttgagag tgtcaggaaa attctctcgg ggactggtga 350

cagtgcccca gcctgagacg gtgcacttct ggccaggctg ggtgcaatga 400

tctgccaggc tgatgggctt cactttggac cccagggatg cctggtcacg 450

cagttgaaga agcatcagat catggttgtg gtcctccaca tcgctgctgt 500

tgtagcaggg gtgtgggatg gactgaacca caggtatttc ttgctctggg 550

ccatctttat tctgtaggct gtggtctccc aggcgtactg tgtatttcgg 600

ttttttacag tgggcagctg taaggaccca gttgccacct acaaggacac 650

cgccacagag tagttgctgg ccctggaaca aggccgcctg ccaaggctgc 700

gaatggggtt ggcactcatg accccccagc accttgtcct cctgtgccct 750

ggagtgtcct gcccaggctc cccccagcaa gagcaggaac atccacgtct 800

tggccgcacg aggtcggggg cgtcccatgg tgaggtcttc cagaatccag 850

ggcattccct gaaacctatc ctccctgaat ttgcctgttg gaggccctta 900

gttttcccac tgaccaatgt gtaattggga ttcaaagcta tacctgaatt 950

tcctacattt ctagtaacag aatttaccaa acatgaactt tctgatggtg 1000

attacgtaac ttctaatttg aagccctttc ctcatctgtg ttttatgtcc 1050

aagaaatgtc tactgatttt gaaagatttg cttcacattc catcactcag 1100

atctaagttt tttctctcaa gtatggactg ttttatatcc tgtaagagct 1150

gaactacctt gacactggag cctttcttgc ttgctatgat tgaaaatcac 1200

atagaaagga ttcctcataa tcctctagga tggtttcatt tacactgtgt 1250

aaattacaag ccatgacccc ctacattctt acattcataa ggtatttctt 1300

ccatttgagt tcggagagac ttggtaagct ctgcctgcta cag 1343

Claims

What is claimed is:

1. Isolated DNA encoding a Tumor Antigen Derived Gene-14 (TADG-14) protein selected from the group consisting of:

(a) isolated DNA having the sequence of SEQ ID No. 6;

(b) isolated DNA which is complementary to the isolated DNA of (a); and

(c) isolated DNA differing from the isolated DNAs of (a) and (b) above in codon sequence due to the degeneracy of the genetic code.

2. The DNA of claim 1, wherein said TADG-14 protein has the amino acid sequence shown in SEQ ID No. 7.

3. A vector capable of expressing the DNA of claim 1, wherein said vector is adapted for expression in a cell and comprises of regulatory elements necessary for expressing said DNA in said cell.

4. The vector of claim 3, wherein said DNA encodes a TADG-14 protein having the amino acid sequence shown in SEQ ID No. 7.

5. The vector of claim 3, wherein said DNA is positioned in reverse orientation relative to said regulatory elements such that TADG-14 antisense DNA is produced.

6. A host cell transfected with the vector of claim 3, wherein said vector expresses a TADG-14 protein.

7. The host cell of claim 6, wherein said cell is selected from group consisting of bacterial cells, mammalian cells, plant cells and insect cells.

8. The host cell of claim 7, wherein said bacterial cell is E. coli.

9. Isolated and purified TADG-14 protein coded for by DNA selected from the group consisting of:

(a) isolated DNA having the sequence of SEQ ID No. 6; and

(b) isolated DNA differing from the isolated DNA of (a) in codon sequence due to the degeneracy of the genetic code, and which encodes a TADG-14 protein.

10. The isolated and purified TADG-14 protein of claim 9 having the amino acid sequence shown in SEQ ID No. 7.

11. A method of detecting TADG-14 mRNA in a biological sample, comprising the steps of:

(a) contacting said sample with a probe specific for TADG-14, wherein said probe comprises of sequence complementary to SEQ ID No. 6; and

(b) detecting hybridization of said probe with TADG-14 mRNA, wherein the presence of hybridization indicates the presence of TADG-14 mRNA.

12. The method of claim 11, wherein said biological sample is selected from the group consisting of blood, interstitial fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells.

13. The method of claim 12, wherein said biological sample is from an individual.

14. The method of claim 13, wherein said individual is suspected of having cancer.

15. A kit for detecting TADG-14 mRNA, comprising:

an oligonucleotide probe specific for TADG-14, wherein said probe comprises of sequence complementary to SEQ ID No. 6.

16. The kit of claim 15, further comprising:

a lable with which to label said probe; and means for detecting said label.

17. A method of detecting TADG-14 protein in a biological ssample, comprising te steps of:

(a) contacting said sample with an antibody specific for TADG-14 or a fragment thereof; and

(b) detecting binding of said antibody to TADG-14 protein in said sample, wherein antibody binding to TADG-14 in said sample indicates the presence of TADG-14 protein in said sample.

18. The method of claim 17, wherein said biological sample is selected from the group consisting of blood, interstitial fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells.

19. The method of claim 18, wherein said biological sample is from an individual.

20. The method of claim 19, wherein said individual is suspected of having cancer.

21. A kit for detecting TADG-14 protein, comprising:

an antibody specific for TADG-14 protein or a fragment thereof.

22. The kit of claim 21, further comprising:

means to detect said antibody.

23. An antibody which is specific for TADG-14 protein or a fragment thereof.

24. A method of screening for compounds that inhibit protease activity of TADG-14, comprising the steps of:

(a) contacting a sample with a compound, wherein said sample comprises TADG-14 protein; and

(b) assaying for TADG-14 protease activity, wherein a decrease in said TADG-14 protease activity in the presence of said compound relative to TADG-14 protease activity in the absence of said compound is indicative of a compound that inhibits TADG-14 protease activity.

25. A method of inhibiting expression of TADG-14 in a cell, comprising the step of introducing the vector of claim 5 into a cell, wherein expression of said vector produces TADG-14 antisense DNA in said cell, wherein said TADG-14 antisense DNA hybridizes to endogenous TADG-14 mRNA and inhibits expression of TADG-14 in said cell.

26. A method of inhibiting a TADG-14 protein in a cell, comprising the step of introducing an antibody specific for TADG-14 protein or a fragment thereof into said cell, wherein binding of said antibody to TADG-14 protein inhibits said TADG-14 protein in said cell.

27. A method of targeted therapy to an individual, comprising the step of:

(a) administering to an individual a compound having a targeting moiety and a therapeutic moiety, wherein said targeting moiety is specific for TADG-14.

28. The method of claim 27, wherein said targeting moiety is selected from the group consisting of an antibody specific for TADG-14 and a ligand or ligand binding domain that binds TADG-14.

29. The method of claim 27, wherein said therapeutic moiety is selected from the group consisting of a radioisotope, a toxin, a chemotherapeutic agent, an immune stimulant and a cytotoxic agent.

30. The method of claim 27, wherein said individual suffers from a cancer selected from the group consisting of ovarian cancer, breast cancer, prostate cancer and colon cancer.

31. A method of diagnosing cancer in an individual, comprising the steps of:

(a) obtaining a biological sample from an individual;

(b) detecting TADG-14 in said sample, wherein the presence of TADG-14 in said sample is indicative of the presence of carcinoma in said individual.

32. The method of claim 31, wherein said biological sample is selected from the group consisting of blood, interstitial fluid, ascites fluid, tumor tissue biopsy and circulating tumor cells.

33. The method of claim 31, wherein said detection of TADG-14 is by means selected from the group consisting of Northern blot, Western blot, PCR, dot blot, ELISA sandwich assay, radioimmunoassay, DNA array chips and flow cytometry.

34. The method of claim 31, wherein said carcinoma is selected from the group consisting of ovarian, breast, colon and prostate cancer.

35. A method of vaccinating an individual against TADG-14 protein, comprising the step of:

inoculating an individual with a TADG-14 protein or fragment thereof that lacks TADG-14 protease activity, wherein said inoculation with said TADG-14 protein or fragment thereof elicits an immune response in said individual, thereby vaccinating said individual against TADG-14.

36. The method of claim 35, wherein said individual is selected from the group consisting of individual with cancer, individual suspected of having cancer and individual at risk of getting cancer.

37. The method of claim 35, wherein the size of said TADG-14 fragment is from 9 amino acids to 20 amino acids.

38. The method of claim 37, wherein said 9 amino acid fragment is selected from the group consisting of SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, 64.

39. A method of producing activated immune cells directed toward TADG-14, comprising the steps of:

exposing immune cells to a TADG-14 protein or fragment thereof that lacks TADG-14 protease activity, wherein said exposure to said TADG-14 protein or fragment thereof activates said immune cells, thereby producing activated immune cells directed toward TADG-14.

40. The method of claim 39, wherein said immune cells are selected from the group consisting of B cells, T cells and dendritic cells.

41. The method of claim 40, wherein said dendritic cells are isolated from an individual prior to exposure to a TADG-14 protein or fragment thereof, wherein said dendritic cells are reintroduced into said individual subsequent to said exposure.

42. The method of claim 41, wherein said individual is selected from the group consisting of individual with cancer, individual suspected of having cancer and individual at risk of getting cancer.

43. The method of claim 39, wherein the size of said TADG-14 fragment is from 9 amino acids to 20 amino acids.

44. The method of claim 43, wherein said 9 amino acid fragment is selected from the group consisting of SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, 64.

45. An immunogenic composition comprising an immunogenic fragment of a TADG-14 protein and an appropriate adjuvant.

46. The immunogenic composition of claim 45, wherein the size of said TADG-14 fragment is from 9 amino acids to 20 amino acids.

47. The immunogenic composition of claim 46, wherein said 9 amino acid fragment is selected from the group consisting of SEQ ID Nos. 17, 18, 41, 42, 47, 48, 53, 56, 64.

48. An oligonucleotide having a sequence of SEQ ID No. 72.

49. A composition comprising the oligonucleotide of claim 48 and a physiologically acceptable carrier.

50. A method of treating a neoplastic state in an individual in need of such treatment, comprising the step of: administering to said individual an effective dose of the composition of claim 49.

51. The method of claim 50, wherein said neoplastic state is selected from the group consisting of ovarian cancer, breast cancer, lung cancer, colon cancer, and prostate cancer in which TADG-14 is overexpressed.